Composition for vaccination

ABSTRACT

The present invention provides novel compositions for transdermal delivery of at least one immunogen to an individual, which compositions comprise (a) said at least one immunogen (b) an occlusion vehicle and (c) an immunogen delivery system in the form of a PosIntro or a cationic ISCOM or comprising at least one saponin and at least one sterol, as well as a process for preparation of the compositions. The invention further provides constructs comprising these compositions. The compositions and constructs may be used in methods for generating an immunological response, methods for treating or preventing a condition of illness and methods for vaccination. The occlusion vehicle is e.g. a pressure sensitive adhesive, such as a hydrocolloid adhesive or a hydrogel adhesive, which may be cross-linked, or the occlusion vehicle may be non-adherent.

FIELD OF THE INVENTION

The present invention relates to novel compositions for transdermaldelivery of at least one immunogen and constructs comprising thesecompositions, a process for preparing the compositions as well as amethod for generating an immunological response, a method for treatingor preventing a condition of illness and a method for vaccination andthe use of an immunogen in such compositions.

BACKGROUND OF THE INVENTION

Transdermal drug delivery (TDD) is a well-known and economicallyimportant and attractive administration route for many drugs, such asnicotine in smoking cessation therapy, isosorbide nitrate or nitroglycerine in angina pectoris therapy and estradiol in hormonereplacement therapy. Transdermal administration typically involves thedelivery of a pharmaceutical agent for percutaneous passage of the drugover the stratum corneum (horny layer) through the epidermis and dermisof the skin or through a mucosal membrane into the systemic circulationof the patient. Transdermal delivery is accomplished by exposing thedrug to the skin or the mucosa of a patient for sustained release of thedrug. In practice, TDD patches including the drug may be adhered to thebody surfaces. TDD administration may also be accomplished byadministration of the drug to the skin as creams, ointments, lotions orthe like.

Transdermal drug delivery involves the use of a vehicle for the drug,which vehicle preferable but not necessarily possesses skin-adheringproperties. A wide number of adhesive systems also called pressuresensitive adhesives systems are generally used, such as silicone,polyurethane, acrylate and polyisobutylene adhesives. These adhesivesare in general non-absorbing, have high occlusivity and high skinadhesion and may accordingly cause incompliance with the patient. Otheradhesive systems with water absorbing capacity, which may have a bettertolerance for the patient, are for instance hydrocolloid adhesives,hydrogel adhesives, cross-linked hydrogel adhesives and the like.

U.S. Pat. No. 4,367,732 describes a hydrocolloid adhesive comprisingstyrene-olefine-styrene block copolymers as matrix molecules compoundedwith various resin tackyfiers and plasticizers being dispersed inhydrocolloid for use in a skin barrier suitable for e.g. bandagepurposes. U.S. Pat. No. 6,153,215 describes the use of hydrocolloidadhesive for delivery of drugs to skin or wounds.

GB patent application No. 2 115 431 discloses a hydrophilic elastomericpressure sensitive adhesive comprising at least one water-insoluble,cross-linked polymer as matrix and additional plasticizers. The adhesiveis useful in various articles, such as bandages and ostomy devices. WO93/00076 discloses a carrier system for drugs comprising sphericalparticles and optionally one or more bio adhesive polymers.

U.S. Pat. No. 5,410,016 discloses hydrogels of polymerised and crosslinked macromer comprising hydrophilic oligomers having biodegradablemonomeric or oligomeric extensions for use as i.a. controlled release ofdrugs or adhesion formation. U.S. Pat. No. 6,410,645 discloses a gelforming macromere comprising at least four covalently linked polymericblocks. The macromers can be cross linked to form a gel on a tissuesurface and are useful in a variety of medical applications includingdrug delivery and tissue coating.

The use of transdermal delivery for vaccination is only scarcelydescribed in literature until now. Furthermore, transdermal delivery ofdrugs is influenced by many different parameters among which is the sizeof the molecules to be delivered. Transdermal delivery is normallyachieved only for quite small molecules of sizes less than 1 nanometer.For instance, Insulin having a molecule size of about 1.5-2 nanometers,is reported to be very complicated to get to pass the skin membrane.

If a transdermal vaccination route could be provided, it would representan important alternative to invasive administration, e.g. by way ofintramuscular, intradermal or subcutaneous injections. Such injectionspresent a range of disadvantages. They may cause stress, pain andirritation, particularly in the case of repeated injections, includingthe risk of infection—or may be poorly tolerated. Besides, untrained orunlicensed persons may not administer injections. Moreover, whenapplying for registration of a vaccine by the authorities, the requireddocumentation of none adverse effects will obviously be more extensivefor vaccines for invasive administration than for transdermaladministration.

Vaccination in the present context is the process by which theapplication of a vaccine to an individual induces an immunologicalresponse in said individual under non-pathogenic conditions.

The transdermal vaccination route which has been described until nowincludes the steps wherein (i) the upper part of the skin, i.e. thestratum corneum, is removed by a mechanical treatment (scraped of) and(ii) the skin is moistened before application of the vaccine oralternatively (iii) the vaccine is absorbed in a gaze pad and applied onthe skin with an ordinary plaster, these techniques is further describedin e.g. Transcutaneous immunization, Glenn G M, Kenney R T. NewGeneration Vaccines, 3rd Ed. Vol. (in press) and in Advances in vaccinedelivery: transcutaneous immunization, Glenn G M, Scharton-Kersten T,and Alving C R. Expert Opinion in Investigational Drugs. Vol. 8 (6)(1999): 797-805, WO 99/43350 and US 2001/0006645 A1.

It is an object of the present invention to provide novel compositionsand a smarter, more simple and safe method for transdermal deliveryunder occlusion of an immunogen to an individual thereby avoiding theadverse effects connected with an invasive administration, such asinjection.

It is a further object of the invention to provide such compositions andmethods, wherein the delivery is accomplished by the use of a deliverysystem, such as Posintro described in WO patent application no.PCT/DK02/00229 or ISCOMs as described in e.g. WO 98/36772, WO 92/06710and WO 98/56420.

It has surprisingly been found that utilizing an occlusion vehicle andthe principles according to the present invention can mediate anenhanced delivery of immunogen and can act as a potentiator of theimmunogen to the immune active cells (often referred to as professionalantigen presenting cells) subsiding beneath mucosal membranes, stratumcorneum or beneath endothelia cell membranes. As the delivery systemand/or adjuvant applied may assume a particulate shape with a mean sizeof about 5 to 50, e.g. most commonly between 10 and 40 nanometers (nm)or even larger, it is an unexpected observation that such relativelylarge particles are capable of demonstrating this enhancing feature.Based on observations regarding delivery of drugs, hormones and proteinsit is generally anticipated that compounds larger than one or a fewnanometers are incapable of penetrating the mentioned cellularmembranes.

SUMMARY OF THE INVENTION

The present invention relates in a first aspect to a composition fortransdermal delivery of at least one immunogen to an individualcomprising

-   -   a) said at least one immunogen    -   b) an occlusion vehicle and    -   c) an immunogen delivery system in the form of a PosIntro or an        ISCOM.

In connection with the present invention the term transdermal deliveryincludes delivery through a skin surface as well as through a mucousmembrane tissue.

In a second aspect the invention relates to a composition fortransdermal delivery of at least one immunogen to an individualcomprising

-   -   a) said at least one immunogen    -   b) an occlusion vehicle in the form of a pressure sensitive        adhesive and    -   c) an immunogen delivery system comprising at least one saponin        and at least one sterol.

As used in connection with the present invention the term occlusion orocclusion vehicle means any covering of skin or mucosal surfaces whichcovering allows a water vapour transmission less than 15000 g/m²×24 hrsin an laboratory test with inverted Paddington cups (BritishPharmacopoeia 1993, addendum 1996, page 1943) with water in contact tothe covering at 37° C. and a relative humidity of 15% in the surroundingenvironment. In praxis considerably lower values will be used, e.g. lessthan 12,000 g/m²×24 hrs, preferably less than 10.000 g/m²×24 hrs, morepreferred less than 6.000 g/m²×24 hrs, or less than 3.000 g/m²×24 hrs,even more preferred less than 1.000 g/m²×24 hrs, or less than 800g/m²×24 hrs, less than 500 g/m²×24 hrs, less than 300 g/m²×24 hrs orless than 200 g/m²×24 hrs. By full occlusion is meant a value by saidtest less than 100 g/m²×24 hrs.

The occlusion may be achieved by any covering in form of e.g. film orfoil material being slightly or non permeable to water. The bestperformance is achieved when such covering is fixed to the body surfaceby adhesives.

The preferred adhesives are able to absorb moisture and retain it in thematrix. This gives the materials the ability to increase the moisturecontent in the skin or the mucosa. The elevated level of moisture in theskin will increase the ability of penetration of a drug. This isdescribed in literature, see e.g. Henning Gjelstrup Kristensen: Almenfarmaci, Institut for Farmaci, Danmarks Farmaceutiske Højskole, 3.udgave (2000), page 332-333. Still further the addition of enhancers tothe formulations of adhesives may add to the penetration rate of thedrug. In the case of mucosal delivery enhancers may not play any role asthe penetration resistance in the endothelial layer is small compared toone of the stratum corneum in skin. On the other hand it may beappropriate to add an enhancer even for mucosal delivery.

A wide number of constructs comprising the compositions of the inventionmay be used. This is illustrated on the drawings on which:

FIG. 1 illustrates a simple monolithic system where the vaccine isapplied topically or homogeneously in a vehicle in this case being apressure sensitive adhesive (2) and where said adhesive is laminated toa cover film (1) at one outer side and a release liner (3) at theopposed side.

FIG. 2 illustrates a reservoir system very similar to that of FIG. 1,but in which the vaccine-containing vehicle (2) is surrounded by anadhesive rim (4). (1) is the cover film and (3) the release liner.

FIG. 3 illustrates a laminate control system identical to the principlesof FIG. 1 except that a thin release-controlling barrier (5) (preferablyporous and pressure sensitive adhesive) is applied between release liner(3) and the vehicle (2).

FIG. 4 illustrates the hybrid between FIG. 2 and FIG. 3. In this systemthe vehicle (2) for the vaccine typically will be liquid (water).

FIG. 5 illustrates a system where the vehicle (2) containing the vaccineis separated from an adhesive (6) intended solely for fixation of theproduct to the human tissue. The vehicle (2) will be applied by gentlepressure at the central indentation in the release liner (3) upon theadhesive part immediately before removal from release liner andsubsequently use. (1) is the cover film. FIG. 6 illustrates a waterreservoir system. In this concept the vehicle (2) containing the vaccineis a lyophilised pad which is separated from the supporting adhesive (6)by being fixed to the release liner (3). Said liner having a centrallyplaced weak point (dotted line) for braking. Further a container (8)with water (7) is welded or adhered to the upper surface of the releaseliner (3). By pressing on the construct the vehicle will adhere to theadhesive (6) in a first step and as a second step the weak point in theliner (dotted line) (3) will break letting the water enter thecompartment with the lyophilised vehicle and by doing so the vehicleturns into a gel.

FIG. 7 illustrates a principle equivalent to FIG. 6 except that that thewater containing compartment (7 and 8) is sealed adjacent to the vehicle(2) still with weak release liner (3) for directing the water into thevehicle.

FIG. 8 illustrates further an equivalent principle to FIG. 6 in whichthe vehicle (2) is fixed at the support adhesive (6). The membrane (10)is sealed to the release liner (3) and the outer film of the watercontaining compartment (8) and divide the compartment from the vehicleand has been made weak for breakage by external pressing at the dottedline. The membrane and the release liner (3) may be one and same throughindentation of the release liner.

FIG. 9 illustrates a two-component packaging for any of above principlesin FIG. 1 through 5. One component will consist of a film (1) and anadhesive support layer (6) at a release liner (3) and the secondcomponent will consist of the vehicle (2) in a pouch (10)

FIG. 10 illustrates the two-component principle equivalent to FIG. 9 incase of water containing constructs as of FIG. 6 through FIG. 8. In thiscase the pouch material (10) is sealed together and has been made weakas illustrated with the dotted line between the vehicle and the watercontainer for directed breakage and water delivery from externalpressing.

The present invention also provides in a third aspect a process for thepreparation of a composition of the invention, comprising the steps ofintroducing the immunogen and the immunogen delivery system, which areoptionally comprised within a vaccine formulation, into the matrix ofthe occlusion vehicle or on its surface by dispersion or soaking in asolution of the vehicle or by applying to its surface, and optionallysterilising and/or drying and/or seal packaging the composition.

In a forth aspect the invention provides a construct comprising acomposition of the invention.

In other aspects the invention provides a method for generating animmunological response in an individual, a method for treating orpreventing a condition of illness in an individual as well as a methodfor vaccination of an individual wherein a composition of the inventionis administered to said individual.

Finally, the invention provides the use of an immunogen for thepreparation of a composition for transdermal delivery of said immunogencomprising an occlusion vehicle.

DETAILED DESCRIPTION OF THE INVENTION

The occlusion vehicle in the composition of the invention is preferablea pressure sensitive adhesive and more preferable an absorbing pressuresensitive adhesive. Such adhesives may especially be found in but arenot limited to the group of hydrocolloid adhesives, hydrogel adhesivesor cross-linked hydrogels. These adhesive systems are further explainedin the following.

Hydrocolloid Adhesive

The hydrocolloid adhesive is a category of adhesives extensively usedfor medical devices intended for fixation to the human body. Theadhesive is a two phase material consisting of a matrix of adhesive inwhich are embedded absorbing particles of hydrophilic polymers theso-called hydrocolloids. The principle is quite flexible as the amountand type of particulate hydrocolloids can be tailor made according tothe intended application and the base adhesive may be aggressive or easyremovable and may further be loaded with suitable enhancers. Also quitemany various types of adhesive raw materials may be used in thehydrocolloid adhesives. Such adhesive raw materials may be compounds orblends including polyacrylates, polyisobutylenes, block copolymershaving styrene end block, polyurethanes, polyvinyl acetate and the likeas commonly known by the person skilled in the art.

Hydrogel Adhesives

In contrast to the hydrocolloid adhesives the hydrogel adhesives arealmost entirely moisture sensitive. This means that the adhesivecontinuously will absorb moisture present in the environment under thechange of properties of the adhesive. In particular, initial absorptionwill lead to good adhesion to moist surfaces. This may for many purposesbe deleterious for its use. However, for one single application andwhere no carrying of weight is required, the adhesive may be a goodalternative, even in moist environments.

The adhesive is classified as a traditional adhesive in which thenormally hydrophobic matrix is changed into a hydrophilic. This isachieved by using a scaffold construction of hydrophilic polymersensuring sufficient cohesion of the adhesive in combination withhydrophilic plasticisers, like polyvalent alcohols and tackifyinghydrophilic oligomers. Further some cross-linking physically orchemically may be introduced into the adhesive for improved cohesion ofthe material. The adhesive is especially suitable for sticking on mucousmembranes.

Hydrogels

The third major group of adhesives for drug delivery is based onhydrogels preferably but not necessarily cross-linked. This groupcorresponds to the hydrogel adhesive to some extent. The essence is,however, that the plasticiser of the hydrogel is exclusively or mainlywater, but it may also include e.g. a polyvalent alcohol like glycerol,polyethylene glycol and polypropylene glycol. Furthermore, the molecularbackbone is preferably chemically cross-linked.

The backbone polymers of the hydrogel may essentially be any hydrophilicpolymer and preferably such polymers that may be suitable forcross-linking. Preferred are polymers from the category of acrylics likeacrylic acid or methacrylates, polyvinyl pyrrolidone, derivatives ofpolyvinyl pyrrolidone and copolymers of such. However, polyethyleneglycols and polyethylene oxide are other often used hydrophilic polymersin cross-linked networks.

The advantage of the hydrogel delivery system may be high solubility ofthe compound being delivered transdermally, and that the high watercontent introduced in the skin from the hydrogel leads to increasedtransdermal penetration. This account when the compound being deliveredtransdermally is a hydrophilic compound.

In one embodiment of the invention the immunogen and the immunogendelivery system are distributed in the occlusion vehicle and thisdistribution is preferably homogenously. This is illustrated in FIG. 1,which shows a “monolithic” system.

The immunogen and the immunogen delivery system may in anotherembodiment also be distributed on the surface of the occlusion vehicle.This will be more efficient and lead to maximal transdermal delivery, asocclusion will lead to decrease in barrier function of the skin and ahigh concentration of immunogen and immunogen delivery system at thesurface of the occlusion vehicle lead to higher instantaneous deliveryof immunogen.

As the composition of the invention must be stable over a long period oftime it may be preferred to separate the immunogen and the immunogendelivery system from adhesive material, thereby avoiding an exchange ofcomponents within the composition. In this embodiment it is also avoidedthat the adhesive leaves residues, such as plasticisers, in thecomposition, and that hydrophilic or hydrophobic immunogens/antigensdiffuses into the total adhesive.

In this embodiment, which may overcome stability issues, the immunogenand the immunogen delivery system may be embedded in a non-adherentvehicle like for instance a dried or lyophilised hydrophilic polymersubstance or in a grease like composition, which potentially may beactivated just before use. This activation may happen by theintroduction of water solution or other appropriate solvent/diluent tothe system. The system will in any case be fixed to the body by acovering, preferably a pressure sensitive adhesive. Accordingly, thecomposition of the invention in another preferred embodiment comprises anon-adherent occlusion vehicle and a secondary adhesive, being separatedfrom the vehicle, for skin fixation.

Another way to overcome the stability issue may be to position a filmbarrier between the vehicle for the vaccine and the secondary adhesive.

The composition may also comprise a rate controlling membrane,preferably of a porous and pressure sensitive material, such as a meshedfilm, open net or the like. The barrier may separate the monolithicsystem from the patient's skin or mucosa as illustrated in FIG. 3.

The occlusion vehicle or the secondary adhesive for skin fixation may beconstituted as a covering, e.g. a pad, a dressing or a patch, or be anyother common covering known in the art.

The composition of the invention may also comprise an enhancer fortransdermal drug delivery. Enhancers in the context of penetrationthrough skin and mucosa are a group of compounds that facilitates thetransport of drugs or vaccines over skin or mucous membranes. In respectto the skin the rate-determining layer for permeation is considered tobe the horny layer (strateum corneum), which is the very outer layer ofthe skin. This must be passed whether the target of the drug or vaccineis the skin per se or the full body. This also indicates that use ofenhancers for improved penetration is of particular importance when skinis the selected barrier.

Typical enhancers used for trans dermal drug delivery are alcohols,amines, phospholipids, fatty acids, surfactants and polyols. Someparticular interesting compounds are low MW-polyethylene glycol,propylene glycol, lauric acid, oleic acid, methyl laurate, ethyl oleate,N-methyl-pyrrolidone, dioctyl adipate and glycerol or low molecularweight derivatives thereof. The requirements to the enhancer will becompatibility with all the present components i.e. the matrix ofadhesive or hydrogel and the vaccine components.

The immunogen and the immunogen delivery system are in yet anotherembodiment separated from each other.

Application of Vaccine and Vaccination

Vaccination in the context of the present invention is the process bywhich the application of a vaccine to an individual induces animmunological response in said individual under non-pathogenicconditions.

Immunogen and Antigen

The composition of the invention may comprise several components, butalways at least one immunogen. Preferably, the immunogen is selected insuch a way that the induced immunological response is directed againstone or more antigens e.g. derived from a pathogenic microorganism.However, the antigens against which the immunological response isdirected may be of non-microbial origin; examples of such antigens aresynthetic antigens, antigens derived from said individual or antigensderived from any species.

Preferably, the induced immunological response confers protection insaid individual against a pathogenic microorganism of which said antigenis part of. Preferably, the induced immunological protection may actupon subsequent exposure of said treated individual to said pathogenicmicroorganism. However, said vaccinated individual may have beeninfected by said pathogenic microorganism before the time of applicationof the vaccine. In this case the protective immunological response maybe functional immediately.

By the term induced immunological response is understood a protectiveresponse, where the conferred immunological response may be completeprotection, leading total elimination of said pathogenic microorganism,however the protection may be partial and only reduce the propagation ofsaid pathogenic microorganism. Furthermore, as the term protection onlyrelates to the reduction or elimination of the state of ill-healthinduced by said pathogenic microorganism viz. the condition to treatand/or the condition to prevent according to the present invention, theprotection may not only be limited to full or partly elimination of saidpathogenic. Example of such a mode of action is a protectiveimmunological response directed against a pathogenic component producedby said pathogenic microorganism during the infection of saidindividual. Examples of such pathogenic components include, but are notlimited to bacterial toxins e.g. tetanus toxin.

In connection with the present invention the immunogens are componentsagainst which it is possible to raise an immune response in anindividual or components, wherein the products of said components cangive rise to an immune response. To obtain the immune response it may benecessary that the immunogen is comprised within a vaccine formulation.

As used in connection with the present invention antigens arecomponents, which can be recognized by an immune response, components,wherein fragments of the components can be recognized by an immuneresponse or components, wherein products of said components or fragmentsof products of said components can be recognized by an immune response.

An immunogen or antigen is in relation to the present invention animmunogen or an antigen preferably derived from a component naturallyassociated with the condition, which is desirable to treat or preventaccording to the present invention. Accordingly, immunogens or antigenwhich are not normally present within cells or associated with cells ofthe treated individual and are desirable to target in order to treat orprevent a specific condition, are to be considered as immunogens orantigens with respect of that particular condition.

Preferably, immunogens or antigens are immunogens or antigens, which arenot derived from the species, which is to be treated. However, inparticular embodiments of the present invention the foreign immunogen orforeign antigens may be derived from the species, which is to betreated. In these embodiments, the immunogen or antigen should notnormally be present or associated with said individuals cells.

In one preferred embodiment, the immunogen and/or antigen is not derivedfrom a human being, such as the immunogen or antigen will not beconsidered as a self-immunogen, when it is administrated to a humanbeing.

In one embodiment of the present invention the immunogen and/or antigencomprises a polypeptide or a peptide, for example the immunogen and/orthe antigen may essentially consist of or consist of a polypeptide or apeptide. The immunogen or antigen may also comprise more than onedifferent polypeptide and/or peptide, such as 2, for example 3, such as4, for example 5, such as 6, for example 7, such as 8, for example 9,such as 10, for example more than 10 different polypeptides.

In some embodiments of the present invention, the immunogen and/orantigen comprises or essentially consists of an organism, preferably amicroorganism or part of an organism, preferably a microorganism andaccordingly the immunogen or antigen may comprise a very large number ofdifferent polypeptides, such as more than 100, for example more than500, such as more than 1000, for example more than 2500.

It is also contained within the present invention that immunogens orantigens may essentially consist of or consist of one or morepolypeptides and/or peptides.

In order to raise an immune response or to enabling an immune responsethe polypeptides may be processed into fragments and the fragments ofthe polypeptides may be the compounds, which are actually recognised bythe immune response.

Polypeptides as used in connection with the present invention mayfurthermore comprise posttranslational modifications, such as forexample phosphorylation, acetylation, methylation, glycosylation or anyother posttranslational modification. In particular, in one embodimentof the present invention the immunogen and/or antigen may comprise aglucosylated polypeptide and/or peptide.

In one preferred embodiment of the present invention the immunogenand/or the antigen comprises a lipopeptide, such as a peptide or apolypeptide chemically linked to a lipid moiety, for example theimmunogen and/or the antigen may essentially consist of or consist of apeptide or a polypeptide chemically linked to a lipid moiety.

In another embodiment of the present invention the immunogen or antigencomprises a nucleic acid sequence, for example the immunogen or antigenmay essentially consist of or consist of a nucleic acid sequence. Theimmunogen or antigen may comprise more than one different nucleic acidsequence, such as 2, for example 3, such as 4, for example 5, such as 6,for example 7, such as 8, for example 9, such as 10, for example morethan 10 different nucleic acid sequences. In some embodiments theimmunogen or antigen may essentially consist of or consist of one ormore nucleic acid sequences.

Preferably, the nucleic acid sequences may encode a polypeptide and/orpeptide. When the nucleic acid sequence encodes a polypeptide and/or apeptide, preferably, the polypeptide and/or peptide and/or fragmentsthereof constitute the compound, which is recognised by the immuneresponse.

Accordingly, the following scenario may take place:

-   -   i) Nucleic acid sequences are targeted to the target cell    -   ii) Nucleic acid sequences are internalised into the target cell    -   iii) Polypeptides and/or peptides are produced within the target        cell    -   iv) Polypeptides and/or peptides and/or fragments thereof are        displayed at the cell surface

Preferably, the polypeptides and/or peptides which are comprised withinthe immunogen or which are encoded by nucleic acid sequences comprisedwithin the immunogen are foreign to the human body. Preferably, thepolypeptides and/or peptides which are comprised within the antigen orwhich are encoded by nucleic acid sequences comprised within the antigenare foreign to the human body.

In yet another embodiment of the present invention the immunogen and orthe antigen comprises a polysaccharide and/or oligosaccharide.Polysaccharides and oligosaccharides comprise at least twomonosaccharides, which may be identical or different. The empiricalformula of a oligosaccharide is (CH₂O)_(n) and range in size fromtrioses (n=3) to heptoses (n=7). Polysaccharides within the scope of thepresent invention may also be branched polysaccharides.

In one preferred embodiment of the present invention the immunogen orantigen is derived from a virus. In another preferred embodiment of thepresent invention the immunogen or antigen is derived from a bacteria.In yet another preferred embodiment of the present invention theimmunogen or antigen is derived from a parasite. In another preferredembodiment of the present invention the immunogen or antigen is derivedfrom a fungus. However, the immunogen or antigen may also comprise amixture of one or more immunogens and/or antigens selected from thegroup consisting of viruses, bacteria, fungi and parasites.

The immunogen and/or antigen may for example be an attenuated virus, anattenuated bacteria, an attenuated fungi or an attenuated parasite.Alternatively, the immunogen or antigen may be an inactivated or akilled microorganism selected from the group consisting of viruses,bacteria, fungi and parasites. Mixtures thereof are also containedwithin the present invention.

Attenuation may for example be accomplished by selecting mutants thathave lost pathogenicity after being cultivated for several generationsin an unnatural host or after mutagenesis or by manipulation of themicroorganism using recombinant DNA techniques. Any other suitablemethod known to the person skilled in the art may also be used forattenuation.

Inactivation and/or killing of micororganisms may be accomplished by anumber of methods, for example heat inactivation, irradiation, chemicalinactivation or any other method known to the person skilled in the art.

The immunogen or antigen may furthermore comprise only a part of amicroorganism selected from the group consisting of viruses, bacteriaand parasites. For example such a part may be a viral capsid, virosomeor a ligation of a bacterial or yeast cell membrane Alternatively, theimmunogen or antigen may only comprise one or more molecules, which havebeen derived from viruses, bacteria, fungi and parasites, such as forexample polypeptides, peptides or nucleic acid sequences.

Furthermore, the immunogen or antigen may comprise molecules such as forexample polypeptides, peptides or nucleic acid sequences, which compriseonly fragments of viral, bacterial, fungi and parasite derivedpolypeptides, peptides or nucleic acid sequences. Such molecules maycomprise more than one fragment. Such molecules may also be chimeric,such as they in addition comprise fragments which are not derived from aviruses, bacteria, fungi and/or parasites or fragments which are derivedfrom another virus, bacteria, fungi and/or parasite.

Additionally, the polypeptides, peptides or nucleic acid sequencesderived from viruses, bacteria, fungi and parasites may have beenmanipulated, for example using recombinant DNA techniques, such as thepolypeptides, peptides or nucleic acid sequences are not the naturallyoccurring molecules, but rather derivatives or mutants thereof. Mutantsinclude mutants, which comprise substitutions, deletions and/oradditions of amino acids or nucleic acids depending on the character ofthe molecule.

Viruses may according to the present invention for example be selectedfrom the group consisting of: Adeno-associated virus, Adenovirus, Avianinfectious bronchitis virus, Baculovirus, Chicken pox, Monkey Pox, AviPox, Corona virus, Cytomegalovirus, Distemper, Enterovirus, Epstein Barrvirus, Feline leukemia virus, Flavivirus, Foot and mouth disease virus,Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E, Herpes species,Herpes simplex, Influenza virus, HIV-1, HIV-2, HTLV 1, Influenza A andB, Kunjin virus, Lassa fever virus, LCMV (lymphocytic choriomeningitisvirus), lentivirus, Measles, Mengo virus, Morbillivirus, Myxovirus,Papilloma virus, Parovirus, Parainfluenza virus, Paramyxovirus,Parvovirus, Poko virus, Polio virus, Polyoma tumour virus, pseudorabies,Rabies virus, Reovirus, Respiratory syncytial virus, retrovirus,rhinovirus, Rinderpest, Rotavirus, Semliki forest virus, Sendai virus,Simian Virus 40, Sindbis virus, SV5, Tick borne encephalitis virus,Togavirus (rubella, yellow fever, dengue fever), Vaccinia virus,Venezuelan equine encephalomyelitis and Vesicular stomatis virus.

Preferably, the virus is selected from the group consisting of influenzaviruses, herpes viruses, morbili viruses, myxo- and paramyxoviruses,flaviviruses, papillomaviruses and hepatitis viruses.

Bacterias may according to the present invention for example be selectedfrom the group consisting of Achromobacter xylosoxidans, Acinetobactercalcoaceticus, preferably A. anitratus, A. haemolyticus, A. alcaligenes,and A. lwoffii, Actinomyces israelii, Aeromonas hydrophilia, Alcaligenesspecies, preferably A. faecalis, A. odorans and A. denitrificans,Arizona hinshawii, Bacillus anthracis, Bacillus cereus, Bacteroidesfragilis, Bacteroides melaninogenicus, Bordetella pertussis, Borreliaburgdorferi, Borrelia recurrentis, Brucella species, preferably B.abortus, B. suis, B. melitensis and B. canis, Calymmatobacteriumgranulomatis, Campylobacter fetus ssp. intestinalis, Campylobacter fetusssp. jejuni, Chlamydia species, preferably C. psittaci and C.trachomatis, Chromobacterium violaceum, Citrobacter species, preferablyC. freundii and C. diversus, Clostridium botulinum, Clostridiumperfringens, Clostridium difficile, Clostridium tetani, Corynebacteriumdiphtheriae, Corynebacterium, preferably C. ulcerans, C. haemolyticumand C. pseudotuberculosis, Coxiella burnetii, Edwardsiella tarda,Eikenella corrodens, Enterobacter, preferably E. cloacae, E. aerogenes,E. hafniae (also named Hafnia alvei) and E. agglomerans, Erysipelothrixrhusiopathiae, Escherichia coli, Flavobacterium meningosepticum,Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis,Haemophilus ducreyi, Haemophilus influenzae, Helicobacter species,Klebsiella species, preferably K. pneumoniae, K. ozaenae og K.rhinoscleromatis, Legionella species, Leptospira interrogans, Listeriamonocytogenes, Moraxella species, preferably M. lacunata and M.osloensis, Mycobacterioum bovis, Mycobacterium leprae, Mycobacteriumtuberculosis, Mycoplasma species, preferably M. pneumoniae, Neisseriagonorrhoeae, Neisseria meningitidis, Nocardia species, preferably N.asteroides and N. brasiliensis, Pasterurella haemolytica, Pasteurellamultocida, Peptococcus magnus, Plesiomonas shigelloides, Pneumococci,Proteus species, preferably P. mirabilis, P. vulgaris, P. rettgeri andP. morganii (also named Providencia rettgeri and Morganella morganiirespectively), Providencia species, preferably P. alcalifaciens, P.stuartii and P. rettgeri (also named Proteus rettgeri), Pseudomonasaeruginosa, Pseudomonas mallei, Pseudomonas pseudomallei, Rickettsia,Rochalimaia henselae, Salmonella species, preferably S. enteridis, S.typhi and S. derby, and most preferably Salmonella species of the typeSalmonella DT104, Serratia species, preferably S. marcescens, Shigelladysenteriae, S. flexneri, S. boydii and S. sonnei, Spirillum minor,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, Streptobacillus moniliformis, Streptococcus, preferablyS. faecalis, S. faecium and S. durans, Streptococcus agalactiae,Streptococcus pneumoniae, Streptococcus pyogenes, Treponema carateum,Treponeam pallidum, Treponema pertenue, preferably T. pallidum,Ureaplasma urealyticum, Vibrio cholerae, Vibrio parahaemolyticus,Yersinia enterocolitica, and Yersinia pestis.

Parasites may according to the present invention for example be selectedfrom the group consisting of Malaria (Plasmodium. falciparum, P. vivax,P. malariae), Schistosomes, Trypanosomes, Leishmania, Filarialnematodes, Trichomoniasis, Sarcosporidiasis, Taenia (T. saginata, T.solium), Leishmania, Toxoplasma gondii, Trichinelosis (Trichinellaspiralis) or Coccidiosis (Eimeria species).

The immunogen and/or antigen could furthermore be derived a fungusselected from the group consisting of Cryptococcus neoformans, Candidaalbicans, Apergillus fumigatus and Coccidioidomycosis.

It is however possible, that the immunogen and/or the antigen is derivedfrom any animal, including for example vertebrates. For example theimmunogen and/or antigen may comprise components derived from or mayessentially consist of the group consisting of ovalbumin, keyhole limpethemocyanin and sperm-whale myoglobulin. Other examples include, but arenot limited to, immunogens and or antigens derived from bee and snakevenoms, and biological active components produced by bloodsuckinganimals, especially insects, for e.g. inhibition of blood coagulation

The immunogen and/or the antigen may in one embodiment comprise a haptenlinked to a carrier molecule, for example the immunogen and/or theantigen may essentially consist of or consist of a hapten linked to acarrier molecule. Alternatively, the product of the immunogen and/or theantigen may comprise or essentially consist of or consist of a haptenlinked to a carrier molecule. Haptens are small chemically definedcompounds that become immunogenic upon conjugation to a carriermolecule, which however are only weakly immunogenic alone. Examples ofhaptens are, but not limited to, bacterial cell-wall components linkedto protein carriers e.g. bacterial toxins or detoxified bacterialtoxins.

The immunogen and/or the antigen may in another embodiment be amultivalent immunogen and/or antigen. Multivalent antigens and/orimmunogens may for example be selected from the group consisting ofT-independent type 2 antigens, synthetic polymers and multimeric peptideantigens. However the immunogen and/or the antigen may also be a anyother multivalent antigen and/or immunogen known to the person skilledin the art.

Preferably, the immunogen and/or the antigen comprises a peptide capableof being presented by MHC molecules, for example the foreign immunogenand/or the foreign antigen may essentially consist of or consist of apeptide capable of being presented by MHC molecules. Alternatively, theproducts of the immunogen and/or the antigen comprises a peptide capableof being presented by MHC molecules, for example the products of theimmunogen and/or the antigen may essentially consist of or consist of apeptide capable of being presented by MHC molecules.

The peptide capable of being presented by MHC molecules may be any suchpeptide known to the person skilled in the art. Preferred examples ofsuch peptides are listed in the following database: “MHCPEP—A databaseof MHC binding peptides (v. 1.3)”, compiled by Brusic (V. Brusic, G.Rudy, A. P. Kyne and L. C. Harrison; MHCPEP, a database of MHC-bindingpeptides: update 1997; Nucleic Acids Research, 1998, Vol. 26, No. 1, pp.368-371).

In addition, the antigen and/or the immunogen may comprise a mixture oftwo or more of the above-mentioned immunogens and antigens.

Preferably, the antigen may be recognised by an immune response raisedagainst the immunogen. Accordingly, the antigen and the immunogen shouldpreferably resemble one another.

More preferably, they resemble one another so that they comprisecompounds, which alone or together with other molecules are capable ofassociating with the same interacting molecule. Alternatively, theycomprise components, wherein fragments of the components alone ortogether with one or more other molecules are capable of associatingwith the same interacting molecule. Interacting molecules within thiscontext are preferably components of the immune system. For exampleinteracting molecules may be selected from the group consisting ofantibodies and T-cell receptors.

In one embodiment of the present invention the antigen and the immunogenare the same, meaning that the immunogen and the antigen are identical.In another embodiment of the present invention the antigen is a fragmentof the immunogen. In yet another embodiment of the present invention theimmunogen is a fragment of the antigen. In a yet further embodiment ofthe present invention the antigen mimics the immunogen.

Immunological Response

The immune response against the immunogen and/or antigen may be presentin the individual prior to application to the treated individual of thecomposition or vaccine formulation according to the present invention.For example such an immune response may have been generated following aninfection of said individual. Accordingly, the method according to thepresent invention includes methods to enhance an existing immunologicalresponse and/or biasing an existing immunological response with respectto current or future mode of action (e.g. cellular vs. humoralresponses), composition (e.g. antibody classes, antibody subclassesetc.), specificity (e.g. recognition of new or different sub-parts ofthe immunogen and or antigen) and/or affinity.

In many cases however, the individual is not immune against theimmunogen and/or antigen prior to application to the treated individualof the composition or vaccine formulation according to the presentinvention. Accordingly, the method according to the present inventionincludes methods of raising an immune response to the immunogen and/orantigen in an individual.

Preferably, the method comprises the steps of:

-   -   i) Immunizing an individual with the immunogen; and    -   ii) Raising an immune response against the immunogen in said        individual

In preferred embodiments the immunogen is administrated to theindividual, internalized into cells of the individual, processed in saidcells and displayed on the surface of said cells.

Immunogenic determinants denote any substance capable of raising animmune response, including a specific antibody response. Any antigenicdeterminant is thus also an immunogenic determinant, but not allimmunogenic determinants are antigenic determinants within the meaningof these terms as used herein. The immunogen may be comprised in avaccine formulation as described herein.

The immunological response, often termed inflammatory response,according to the present invention may be mediated by any components ofthe immune system of the treated individual.

The immune system may exhibit both specific and non-specific immunity(Klein, J., et al., Immunology (2nd), Blackwell Science Inc., Boston(1997)). Generally, B and T lymphocytes, which display specificreceptors on their cell surface for a given antigen, produce specificimmunity. The immune system may respond to different antigens in twoways: 1) humoral-mediated immunity, which includes B cell stimulationand production of antibodies or immunoglobulins [other cells are alsoinvolved in the generation of an antibody response, e.g.antigen-presenting cells (APCs; including macrophages), and helper Tcells (Th1 and Th2)], and 2) cell-mediated immunity (CMI), whichgenerally involves T cells including cytotoxic T lymphocytes (CTLs),although other cells are also involved in the generation of a CTLresponse (e.g., Th1 and/or Th2 cells and APCs).

Non-specific immunity encompasses various cells and mechanisms such asphagocytosis (the engulfing of foreign particles or antigens) bymacrophages or granulocytes, and natural killer (NK) cell activity,among others.

Non-specific immunity relies on mechanisms less evolutionarily advanced(e.g., phagocytosis, which is an important host defense mechanism) anddoes not display the acquired nature of specificity and memory,hallmarks of a specific immune response. Nonspecific immunity is moreinnate to vertebrate systems. In addition, cells involved in nonspecificimmunity interact in important ways with B and T cells to produce animmune response.

The key differences between specific and nonspecific immunity are basedupon B and T cell specificity. These cells predominantly acquire theirresponsiveness after activation with a specific antigen and havemechanisms to display memory in the event of future exposure to thatspecific antigen. As a result, vaccination (involving specificity andmemory) is an effective protocol to protect against harmful pathogens.

The cell-mediated may be a cytolytic process or comprise a cytolyticprocess. Preferably, however the cytotoxic and/or inflammatory responseaccording to the present invention is mediated by cytotoxic T-cells.Such cytotoxic T-cells preferably express T-cell receptors that canassociate with the antigen or fragments of the antigen or products ofthe antigen or fragments of products of the antigen according to thepresent invention.

However, the cytotoxic and/or inflammatory response may also be mediatedby natural killer cells. In addition the cytotoxic and/or inflammatoryresponse may for example be mediated by neutrophils or the cytotoxicand/or inflammatory response may be mediated eosinophils.

Furthermore, the cytotoxic and/or inflammatory response may be mediatedby antibody-dependent cell-mediated cytotoxicity (ADCC) mechanisms. Suchmechanisms preferably involve antibodies that can associate with theantigen or fragments of the antigen or products of the antigen orfragments of products of the antigen according to the present invention.

In one embodiment of the present invention, the cytotoxic and/orinflammatory response may be mediated by the innate immune system. Forexample the cytotoxic and/or inflammatory response may be mediated bythe complement cascade. For example, the cytotoxic and/or inflammatoryresponse may be mediated by the process of opsonisation by antibodies.For example, the cytotoxic and/or inflammatory response may be mediatedby the process of opsonisation by one ore more components of thecomplement system.

In particular, the cytotoxic and/or inflammatory response may bemediated by of opsonisation of antibodies on the cellular surface oftarget cells by the classical complement pathway or the cytotoxic and/orinflammatory response may be initiated directly by the activation ofcomplement factors, the alternative complement pathway.

Following the activation of one or both complement pathways the terminalmembrane-attached complex may be formed, the target cell may be lysedand eliminated by said complex. In one preferred embodiment of thepresent invention the cytolytic process may result from the formation ofa membrane-attack complex.

Antibodies that may activate the complement pathways according to thepresent invention, may for example be antibodies selected from the groupconsisting of IgG1, IgG2, IgG3 and IgM antibody isotypes.

Antibody-dependent cell-mediated cytotoxicity (ADCC) mechanisms maycomprise other anti-cellular effectors than the complement pathways. Forexample antibodies bound to targeted cell may direct cytolyticactivities of eosinophiles against the targeted cells. In such anembodiment the antibodies preferably are IgE.

In addition, the cytotoxic and/or inflammatory response may be mediatedby a combination of two or more mechanisms mentioned herein above. Forexample it may be mediated by two or more selected from the groupconsisting of cytotoxic T-cells, natural killer cells, neutrophils,eosinophils, antibody-dependent cell-mediated cytotoxicity and cytolyticmechanisms.

Immunogen Delivery System

The immunogen delivery system according to the present invention may beany immunogen delivery targeting system capable of being targeted totarget cells of an individual, wherein the target cells are desirable totarget and/or eliminate. A number of different immunogen deliverytargeting systems are known to the person skilled in the art and asuitable immunogen delivery targeting system may be selected accordingto the specific need.

In a preferred embodiment the immunogen and the immunogen deliverysystem is comprised within a vaccine formulation. Any vaccineformulation known to the person skilled in the art may be used inconnection with the present invention.

In one embodiment of the present invention the immunogen delivery systemis a cationic ISCOM.

PosIntros or ISCOMS may for example be any of the compounds described inthe pending WO patent application no. PCT/DK02/00229, which is herebyincorporated by reference in its entirety.

PosIntros within the scope of the present invention are complexescomprising:

-   -   i) at least one first sterol and/or at least one second sterol,        wherein the at least one second sterol is capable of contacting        a foreign antigen, preferably a nucleic acid by means of an        interaction selected from an electrostatic interaction and a        hydrophobic interaction, and wherein the at least one first        sterol and/or the at least one second sterol is capable of        forming a complex with at least one first saponin and/or at        least one second saponin, and    -   ii) at least one first saponin and/or at least one second        saponin,        wherein the at least one second saponin is capable of contacting        a genetic determinant by means of an interaction selected from        an electrostatic interaction and a hydrophobic interaction, and        wherein the at least one first saponin and/or the at least one        second saponin is capable of forming a complex with at least one        first sterol and/or at least one second sterol, and optionally    -   iii) at least one contacting group for contacting a genetic        determinant by means of an interaction selected from an        electrostatic interaction and a hydrophobic interaction,    -   with the proviso that the at least one contacting group is        present when no second sterol is present in the complex and        further optionally    -   i) at least one lipophilic moiety.

PosIntros according to the present invention may in one preferredembodiment adopt a micro-particle structure in the form of a cage-likematrix similar to that known as an immune stimulating complex (iscom).Beside iscom structures, the interaction between sterols and saponinshave been reported to result in a variety of different structuralentities, including entities such as e.g. lattices, honeycombes, rods,and amorphic particles, all of which structural entities are covered bythe present invention.

In another embodiment the immunogen delivery system comprises aliposome. A liposome within the meaning of the present invention isgenerally spherical or spheroidal cluster or aggregate of amphipathiccompounds, including lipophilic moieties, typically in the form of oneor more concentric layers, for example, monolayers, bilayers ormulti-layers. They may also be referred to herein as lipid vesicles. Theliposomes may be formulated, for example, from ionic lipids and/ornon-ionic lipids. Liposomes formulated from non-ionic lipids may bereferred to as niosomes. Liposomes formulated, at least in part, fromcationic lipids or anionic lipids may be referred to as cochleates.

The liposomes may be prepared e.g. as described by Lipford and Wagner(1994) in Vaccine, vol. 12, no. 1, p. 73-80, incorporated herein byreference. General liposomal preparatory techniques which may be adaptedfor use in the preparation of liposome compositions pertaining to thepresent invention are discussed, for example, in U.S. Pat. Nos.4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and4,921,706; U.K. Patent Application GB 2193095A; InternationalApplication Serial Nos. PCT/US85/01161 and PCT/US89/05040; Mayer et al.,Biochimica et Biophysica Acta, 858:161-168 (1986); Hope et al.,Biochimica et Biophysica Acta, 812:55-65 (1985); Mayhew et al., Methodsin Enzymology, 149:64-77 (1987); Mayhew et al., Biochimica et BiophysicaActa, 755:169-74 (1984); Cheng et al, Investigative Radiology, 22:47-55(1987); and Liposome Technology, Gregoriadis, G., ed., Vol. I, pp.29-31, 51-67 and 79-108 (CRC Press Inc., Boca Raton, Fla. 1984), thedisclosures of each of which are hereby incorporated by referenceherein.

Accordingly, the liposome compositions may be prepared using any one ofa variety of conventional liposomal preparatory techniques which will beapparent to one skilled in the art, including, for example, solventdialysis, French press, extrusion (with or without freeze-thaw), reversephase evaporation, simple freeze-thaw, sonication, chelate dialysis,homogenization, solvent infusion, microemulsification, spontaneousformation, solvent vaporization, solvent dialysis, French pressure celltechnique, controlled detergent dialysis, and others, each involving thepreparation of the compositions in various fashions. See, e.g., Maddenet al., Chemistry and Physics of Lipids, 53:37-46 (1990), the disclosureof which is hereby incorporated herein by reference.

Suitable freeze-thaw techniques are described, for example, in WOapplication no. PCT/US89/05040, filed Nov. 8, 1989, the disclosure ofwhich is hereby incorporated herein by reference in its entirety.Methods. which involve freeze-thaw techniques are preferred inconnection with the preparation of liposomes. Preparation of theliposomes may be carried out in a solution, such as an aqueous salinesolution, aqueous phosphate buffer solution, or sterile water. Theliposomes may also be prepared by various processes which involveshaking or vortexing, which may be achieved, for example, by the use ofa mechanical shaking device, such as a Wig-L-Bug™ (Crescent Dental,Lyons, Ill.), a Mixomat (Degussa AG Frankfurt, Germany), a Capmix (EspeFabrik Pharmazeutischer Praeparate GMBH & Co., Seefeld, Oberay Germany),a Silamat Plus (Vivadent, Lechtenstein), or a Vibros (Quayle Dental,Sussex, England). Conventional microemulsification equipment, such as aMicrofluidizer™ (Microfluidics, Woburn, Mass.) may also be used.

In one embodiment of the invention the immunogen delivery systemcomprises a biodegradable microsphere. In another embodiment theimmunogen delivery system comprises an encapsulation system. In onepreferred embodiment the immunogen delivery system comprises acochleate. In yet another embodiment the immunogen delivery systemcomprises a nanoparticle. In a still further embodiment the immunogendelivery system comprises a hydrogel. In an even still furtherembodiment the immunogen delivery system comprises a microcrystal.

The composition and vaccine formulation may comprise more than oneimmunogen or antigen, such as 2, for example 3, such as 4, for example5, such as more than 5 different antigens. The immunogens and antigensmay be selected from the group of immunogens and antigens describedherein above.

The compositions according to the invention may contain conventionallynon-toxic pharmaceutically acceptable carriers and excipients. Thepharmaceutically acceptable excipients may include adjuvants,penetration adjuvants, emulsifying agents, antioxidants, bufferingagents, preservatives, humectants, chelating agents, gel forming agentsand the like and are all selected in accordance with conventionalpharmaceutical practice in a manner understood by the persons skilled inthe art of formulating pharmaceuticals.

Preferably, the composition and vaccine formulation according to thepresent invention furthermore comprises an adjuvant. The vaccineformulation according to the present invention may furthermore comprisea carrier. The carrier or adjuvant could be any carrier or adjuvantknown in the art including functional equivalents thereof. Functionallyequivalent carriers are capable of presenting the same antigen inessentially the same steric conformation when used under similarconditions. Functionally equivalent adjuvants are capable of providingsimilar increases in the efficacy of the composition when used undersimilar conditions.

Preferably, said formulations comprise potent, nontoxic adjuvants thatwill enhance and/or modulate the immunogenicity of immunogenicdeterminants including antigenic determinants including haptenicdeterminants represent one group of preferred adjuvants. In addition,such adjuvants preferably also elicit an earlier, more potent, or moreprolonged immune response. Such an adjuvant would also be useful incases where an immunogen supply is limited or is costly to produce.

Adjuvants pertaining to the present invention may be grouped accordingto their origin, be it mineral, bacterial, plant, synthetic, or hostproduct. The first group under this classification is the mineraladjuvants, such as aluminium compounds. Antigens precipitated withaluminium salts or antigens mixed with or adsorbed to performedaluminium compounds have been used extensively to augment immuneresponses in animals and humans. Aluminium particles have beendemonstrated in regional lymph nodes of rabbits seven days followingimmunisation, and it may be that another significant function is todirect antigen to T cell containing areas in the nodes themselves.Adjuvant potency has been shown to correlate with intimation of thedraining lymph nodes. While many studies have confirmed that antigensadministered with aluminium salts lead to increased humoral immunity,cell mediated immunity appears to be only slightly increased, asmeasured by delayed-type hypersensitivity. Aluminium hydroxide has alsobeen described as activating the complement pathway. This mechanism mayplay a role in the local inflammatory response as well as immunoglobulinproduction and B cell memory. Furthermore, aluminium hydroxide canprotect the antigen from rapid catabolism. Primarily because of theirexcellent record of safety, aluminum compounds are presently the onlyadjuvants used in humans.

Another large group of adjuvants is those of bacterial origin. Adjuvantswith bacterial origins can be purified and synthesized (e.g. muramyldipeptides, lipid A) and host mediators have been cloned (Interleukin 1and 2). The last decade has brought significant progress in the chemicalpurification of several adjuvants of active components of bacterialorigin: Bordetella pertussis, Mycobacterium tuberculosis,lipopolysaccharide, Freund's Complete Adjuvant (FCA) and Freund'sIncomplete Adjuvant (Difco Laboratories, Detroit, Mich.) and MerckAdjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Additionallysuitable adjuvants in accordance with the present invention are e.g.Titermax Classical adjuvant (SIGMA-ALDRICH), ISCOMS, Quil A, ALUN, seeU.S. Pat. Nos. 58,767 and 5,554,372, Lipid A derivatives, choleratoxinderivatives, HSP derivatives, LPS derivatives, synthetic peptidematrixes, GMDP, and other as well as combined with immunostimulants(U.S. Pat. No. 5,876,735).

B. pertussis is of interest as an adjuvant in the context of the presentinvention due to its ability to modulate cell-mediated immunity throughaction on T-lymphocyte populations. For lipopolysaccharide and Freund'sComplete Adjuvant, adjuvant active moieties have been identified andsynthesized which permit study of structure-function relationships.These are also considered for inclusion in immunogenic compositionsaccording to the present invention.

Lipopolysaccharide and its various derivatives, including lipid A, havebeen found to be powerful adjuvants in combination with liposomes orother lipid emulsions. It is not yet certain whether derivatives withsufficiently low toxicity for general use in humans can be produced.Freund's Complete Adjuvant is the standard in most experimental studies.

Mineral oil may be added to vaccine formulation in order to protect theantigen from rapid catabolism.

Many other types of materials can be used as adjuvants in immunogeniccompositions according to the present invention. They include plantproducts such as saponin, animal products such as chitin and numeroussynthetic chemicals.

Adjuvants according to the present invention can also been categorizedby their proposed mechanisms of action. This type of classification isnecessarily somewhat arbitrary because most adjuvants appear to functionby more than one mechanism. Adjuvants may act through antigenlocalization and delivery, or by direct effects on cells making up theimmune system, such as macrophages and lymphocytes. Another mechanism bywhich adjuvants according to the invention enhance the immune responseis by creation of an antigen depot. This appears to contribute to theadjuvant activity of aluminum compounds, oil emulsions, liposomes, andsynthetic polymers. The adjuvant activity of lipopolysaccharides andmuramyl dipeptides appears to be mainly mediated through activation ofthe macrophage, whereas B. pertussis affects both macrophages andlymphocytes. Further examples of adjuvants that may be useful whenincorporated into immunogenic compositions according to the presentinvention are described in U.S. Pat. No. 5,554,372.

In one preferred embodiment, adjuvants according to the presentinvention are selected from the group consisting of aluminium compounds,Freunds incomplete adjuvant, Titermax classical adjuvant and oilemulsions.

There is also provided an embodiment of the present invention whereinthe composition or vaccine formulation further comprises a carrier. Thecarrier may be present independently of an adjuvant. The purpose ofconjugation and/or co-immunisation of an antigen and a carrier can bee.g. to increase the molecular weight of the antigen in order toincrease the activity or immunogenicity of the antigen, to conferstability to the antigen, to increase the biological activity of thedeterminant, or to increase its serum half-life. The carrier protein maybe any conventional carrier including any protein suitable forpresenting antigens. Conventional carrier proteins include, but are notlimited to, keyhole limpet hemocyanin, serum proteins such astransferrin, bovine serum albumin, or human serum albumin, an ovalbumin,immunoglobulins, or hormones, such as insulin.

The composition or vaccine formulation according to the presentinvention may furthermore comprise a biological active component. Abiological active component may be any component, which directly orindirectly can influence the immune response of an individual.Preferably, the biological active component is selected from the groupconsisting of cytokines and chemokines.

Cytokines may for example be selected from the group consisting of IL-2,IL-4, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-γ, IFN-α, GM-CSF, C-CSF.The compositions of the invention may be manufactured in various ways.

For monolithic use of the adhesive the the immunogen and the immunogendelivery system optionally comprised within a vaccine formulation may beintroduced into the matrix of the adhesive to a homogeneous distributionby spraying this dispersion onto the particular hydrocolloids beforethese are mixed within the adhesive matrix. During the spraying thehydrocolloid particles shall be mixed and rotated to secure an evendistribution of the immunogen and its delivery system. In a second stepthe hydrocolloid is mixed at relatively low temperatures into thethermoplastic molten adhesive and in a third step the final blend iscoated in the preferred thickness by a conventional coating technique.

In respect to hydrocolloid adhesives the release rate is very slow fromsuch homogeneously distributed immunogen or vaccine components. Forcross-linked hydrogels containing homogeneously distributedimmunogen/immunogen delivery system or vaccine components the releaserate appears to be faster. When the immunogen delivery system isPosIntros, i.e. nanoparticles, it is preferred to apply these to thesurface of the adhesives. This is preferably done from an aqueousdispersion and results in an isotropic material with inhomogeneousdistribution of the PosIntros. The surface with the high content ofPosIntros is intended to be the releasing side of the material.

In other embodiments the immunogen delivery system and the immunogen areused in dry state. This may be obtained by simple drying orlyophilisation of these components. Preferably however a processassistant agent like a hydrophilic polymer is used for creation ofbetter bulk properties. Such agent may be polysaccharides and cellulosematerial or synthetic polymers like polyvinyl pyrrolidone and polyvinylalcohol but are not limited to those. Drying solutions may result inbrittle pads or porous structures depending on type of process. Betterproperties may be achieved by adding plasticiser and/or appropriateenhancers for trans dermal drug delivery. The immunogen and the deliverysystem may also be soaked into porous cross-linked material and in asecond step be dried.

The finished products will in general be sterile. This impliesappropriately sealed packaging chosen according to the selected methodof sterilization. The preferred methods of sterilization are beta orgamma radiation. However, autoclaving may be used for hydrogels andethylene oxide sterilization or plasma sterilization for hydrocolloidand hydrogel adhesives. Autoclaving is the only method that isinappropriate for lyophilised or otherwise dry pads based on hydrophilicpolymers.

The construct of the invention has in one embodiment more than onecompartment and in another embodiment at least two compartments, whereina first compartment comprises a lyophilised pad comprising the immunogenand the immunogen delivery system and a second compartment comprisesaqueous solution.

Film and Release Liners

For practical use the construct of the invention will comprise a coverfilm and/or a release liner. The covering, e.g. the patch, pad ordressing containing the immunogen or vaccine components comprises abarrier to the side not in contact with the tissue. Such barrier may bea film of a polymeric material like polyurethane, polyethylene,polyamide, polyester, polyvinyl alcohol and the like but should not berestricted to such. The barrier may be of metal foil or even thinceramics. Accordingly, the choice of the barrier film will not becritical to the invention. Typical the requirement to said barriermaterial would be flexibility and conformability, for which reason thinpolymeric films are preferred.

For protection of the opposite side of the patch release liners based one.g. silicone and the like will be preferred, but other materials may beused. They may be based on sheets of paper or plastic film. In case ofcross-linked hydrogels, plastic films are preferred and siliconisingwill normally not be required.

Other films, which will be suitable according to the invention, forinstance for constructs comprising two component or for constructscomprising a water containing compartment may be quite ordinary for theperson skilled in the art; and preferably multilaminate barrier filmsfrom plastics are used.

The conditions to be treated and the conditions to be prevented by thecompositions of the present invention are conditions of illness of anindividual where the progress of a disease can be reduced or eliminatedby a recognition of the disease-causing agent by said individuals ownimmune system after specific induction of a immunological response insaid individual.

Typically, the condition is, but not limited to, a disease caused byinfection of said individual by a pathogenic microorganism.

EXAMPLES Example 1

Preparation of Vaccine (Immunogen/Immunogen Delivery System)(Experimental Tetanus Vaccine)

Mega 10 (N-Decanoyl-N-methylglucamide) was purchased from Sigma-Aldrich,St. Louis, Mo. and used as a 20% stock solution. Cholesterol,DC-cholesterol (Sigma-Aldrich, St. Louis, Mo.) and phophatidylcholine(Epikuron 200S, Lucas Meyer Gmbh, Germany) was dissolved in 20% Mega 10at a concentration of 1% w/v with respect to each component and storedat −20° C. Quil A saponin was obtained form Superfos Biosector,Frederikssund, Denmark. Quil A was dissolved (15 mg/ml) in Milli-Q waterand filtered to sterility and stored in solution at −20° C. until used.

Tetanus-PosIntro™ was prepared by combining Quil A,cholesterol/DC-cholesterol and phophatidylcholine with Tetanus toxoid(List Biological Laboratories, Inc.). PosIntro™ micro-particles wereallowed to form for 4 hours with stirring at 35° C. followed by dialysisagainst phosphate buffered saline (pH 7.2) in Slide-A-Lyzer® cassettes(Pierce, Rockford, Ill.) or dialysis tubing (Visking, London, UK), MWcut-off 10,000. The concentration in the reaction mixture of Quil A was1.1 mg/ml and the concentration of cholesterol, DC-cholesterol andphophatidylcholine was 0.03% w/v. The final concentration of Mega 10 was4% w/v unless otherwise stated. The concentration of Tetanus toxoid inthe reaction mixture ranged from 0.1 to 0.5 mg/ml, depending on theexperiments.

Example 2

Purification of Tetanus-PosIntro™.

Sucrose gradients were prepared from 25% (w/v) sucrose in phosphatebuffered saline (PBS) pH 7.2. Polyallomer™ centrifuge tubes (size 13×51mm, Beckmann Instruments) were filled with 4.5 ml sucrose solution andstored at −20° C. Upon usage the required number of tubes was allowed tothaw slowly at 4° C., typically overnight, whereby the gradient formed.Gradients were allowed to equilibrate at room temperature for at least 1hour before Tetanus-PosIntro™-preparations were applied in a volume of0.5 ml or less. Centrifugation was performed in a Beckmann Instrumentsrotor type SW55Ti at 20° C., 50,000rpm for 3 hours. Intactmicro-particles were colleted through a diode array UV-detector (PerkinElmer) and monitored at 210 nm.

Purified Tetanus-PosIntro™ micro particles were dialysed first againstphosphate buffered saline (pH 7.2) in Slide-A-Lyzer® cassettes (Pierce,Rockford, Ill.) in order to remove sucrose. The vaccine preparation wasfurther up-concentrated by reverse dialysis against Slide-A-LyzerConcentrating Solution™ (Pierce, Rockford, Ill.). The selected liquidvaccine formulation contained Tetanus toxoid embedded into PosIntro™micro-particles with sizes of 35-50 nm as visualized by transmissionelectron microscopy. The concentration of Tetanus toxoid was 4.2 mg/mlwhereas the concentration of Quil A was 12 mg/ml.

Example 3

Amino Acid Analysis

The Pico-Tag System® from Waters Corporation was used for determinationof protein content of PosIntro™ micro-particles. Hydrolysis wasperformed in a Pico-Tag workstation at 150° C. for 1 hour using 6N HCland phenol, followed by derivatization with phenylisothiocyanale (PITC)according to the Pico-Tag protocol. In order to obtain quantitativemeasurement the PosIntro™-structure was disrupted by treatment with 1:1dichloromethane (DCM) and acetonitril (ACN) for 10 min at roomtemperature after which solvent/diluent was evaporated in vacuum

Example 4

Hydrocolloid Adhesive as Vehicle for Vaccine (Immunogen and DeliverySystem)

The hydrocolloid vehicle is produced as follows: In a Z-blade mixer isintroduced 45 g of a tackyfier resin (Arkon P90, Arakawa) at 140 ° C.After 5 minutes mixing 32.5 g of a block copolymer Styrene-Isopreneelastomer (Kraton TR 1107, Shell) and 5 g dioctyl adipate are added.After further 10 minutes further 7 g dioctyl adipate is added and themixture is blended until homogeneous. Finally 30 g of carboxy methylcellulose (Aquasorb, Hercules) is added. The mixture is coated by hotmelt coating in a thickness of 0.5 mm at a film of polyurethane (30 μm)coated at a silicone paper.

A solution of the vaccine prepared as described in example (vaccineequivalent to 0.100 mg/cm²) is applied evenly to circular areas of 15 mmat the surface of the adhesive before drying and followed by laminatingwith a release-liner. Finally patches are die-cut from the adhesive inround patches with the vaccine area in the centre and in diameters of 30mm.

The patches are packed individually and sterilized by electron beamradiation.

Example 5

Hydrocolloid Adhesive as Vehicle for Vaccine

A hydrocolloid adhesive is produced as described in example 4 exceptthat the dioctyl adipate is replaced as follows: the first addition ofadipate is replaced with 10 g paraffin oil and the second is replaced to15 g paraffin oil. The homogeneous mass is hot melt coated in athickness of 400 μm at a polyurethane film laminated to a release paper.The solution of vaccine prepared as described in example 1 is sprayedevenly to the adhesive's open surface in circular areas of 10 mm and inan amount equalling 0.100 mg/cm².

After drying a release liner is laminated to the open surface and thefinal patches are die-cut from the laminate in sizes of 30 mm having thevaccine area in the center. Eventually the patches are packed andsterilized with gamma radiation at 30 kGy.

Example 6

Cross-Linked Hydrogel as Vehicle for Vaccine

A homogeneous blend of Polyvinyl Pyrrolidone (Plasdone K 120, ISP) 15 g,polyethylene glycol 5 g and demineralised water 80 g is poured into adish in a layer thickness of 0.5 mm. An open structured non-woven ofpolyester is placed on top of this layer and an additional layer of 0.5mm of the homogeneous blend is added. Finally a layer of 30 μm thickpolyurethane film is applied on top of the construct. The dish is thenbeta-radiated with 30 kGy for cross-linking.

After cross-linking the construct is removed from the dish and inverted.On the new top surface opposed to the polyurethane film a layer ofvaccine prepared as described in example 1 is sprayed in a concentrationof 0.050 mg/cm².

The finished construct is die-cut into patches and packed in sealedpackaging of metallized barrier laminate film.

Example 7

Cross-Linked Hydrogel as Vehicle for Vaccine

A homogenized blend of PVP K 90 (Plasdone), glycerol and water in ratios20/15/65 is further added 1.0 part of ethoxylated (4)pentaerythritoltetraacrylate (from Sartomer) and a cross-linking photoinitiater(Darocure 1173 from CIBA) in an amount 0.5%. To the blend is furtherintroduced vaccine prepared as described in example 1 corresponding to0.05% determined by dry matter.

The blend is coated at a polyethylene film (barrier film) in a thicknessof 1 mm on top of the coating a polyurethane film in a thickness of 25μm is laminated. The coated and laminated material is in a followingstep cured with UV-light (Fusion UV Systems, Inc) for 60 seconds.

The laminate is die-cut to patches and packed.

Example 8

Lyophilised Cross-Linked Hydrogel as Vehicle for Vaccine

The patch as produced in example 4 is frozen (−40 ° C.) and lyophilised(24 hrs) in standard laboratory equipment. The resulting porous pad isapplied to the center of a hydrocolloid adhesive patch without vaccineas of example 1 leaving at least 5 mm free adhesive surrounding. Saidpatch will at one outer surface be covered with a polyurethane film andat the other a release liner.

The patch is packed individually in airtight pouch and sterilized bybeta radiation.

Example 9

Lyophilised Cellulose Derivative as Vehicle for Vaccine

Vaccine (0.025%) prepared as described in example 1 is mixed with a 5%aqueous solution of hydroxyethyl cellulose (Natrosol 250 M, pharm,Hercules Inc) and 0.5% PEG 300 and further 0.1% glycerol. The resultingblend is casted into wells of 15 mm in diameter in a layer of 0.5 mm inthickness. The tray with the wells and the casting is transferred to afreezer (−20 ° C.) and after frozen (24 hrs) the tray is transferredinto a freeze dryer for lyophilization.

The pads of the wells are transferred to centres of die-cut adhesivepatches based on a hydrocolloid adhesive as of example 8. The adhesivehas an outer rim of 0.5 cm of free adhesive. Finally a release liner isapplied to top of the patch.

The patch is packed in airtight packaging and sterilized by betaradiation. In use the central pad is wetted by water for improvingrelease of the vaccine.

Example 10

Lyophilised Cellulose Derivative as Vehicle for Vaccine Packed withWater Container

The patch as described in example 9 is packed in a construct having twocompartments. The first compartment contains the patch with releaseliner but in which case the release liner does not cover the centralarea with the lyophilised pad containing the vaccine. The secondcompartment is a container for water. The two compartments are sealedtogether in a way that the separating cover film comprises a breakableindentation between the compartments. When the water compartment ispressed upon the indentation is broken and water will diffuse into thelyophilised pad.

The breaking of the seal will be done just before use of the patch.

Example 11

Hydrogel Adhesive as Vehicle for Vaccine

A hydrogel adhesive is produced as follows. 100 g PVP K-90 is dissolvedin 400 g of ethanol. The solution is further added 20 g propylene glycoland 60 g of ordinary dry potato starch and additional 40 g of waterunder heating. When the mixture appears homogeneous it is coated at afilm backing of polyvinyl alcohol (50 μm) in a layer of 1 mm and dried.The resulting film is in the range of 300 μm.

In a second step the vaccine prepared as described in example 1 issprayed in a concentration corresponding to 0.05 mg/cm². The product isdried and die-cut into patches.

Example 12

Hydrogel Adhesive as Vehicle for Vaccine

A hydrogel adhesive is produced according to hot melt mixing principles.30 g PVP K 25, 4.0 g PVA (Elvanol HV from Du Pont de Nemours & Co) and36 g glycerol is blended in a z-blade mixer at 100° C. When the mixtureappears homogeneous further 30 g de-mineralised water is added. Thefinal blend is pressed into a thickness of 300 μm between a releasepaper and a polyethylene film of 50 μm.

Example 13

Hydrogel Adhesive as Vehicle for Vaccine

20 g of polyvinyl-pyrrolidone (PVP K90) was mixed with 4 g ofpolyethylene-glycol dimethacrylat 1000 (PEG-DMA 1000) (cross-linkingagent) and 1 g sodium peroxiddisulfate (NaPS) (photoinitiator) in 75 gof 0.1 M citric acid/citrate buffer pH 6.0. Propylene glycol could beadded as a plastiziser (See Table of composition below).

The polymer solution was dispensed into a suitable mold in which thethickness could be varied from 0.5-5 mm thickness. If backing wasrequired the polymer solution was dispensed onto the backing filmcovering the bottom of the mold.

The polymer solution was then cured under UV-light. The hydrogel wasUV-cured under a single UV-lamp (specifications: 200 W/cm, microwavepowered “D”-spectral type lamp with a conveyor speed of 0.4 m/min). Asheet hydrogel was obtained from which hydrogels of the desired diametercould be cut. TABLE 1 Hydrogel composition and dimension Type ofHydrogel Backing Thickness Diameter Hydrogel 1: PVP K90 20 g nonePEG-DMA 4 g 1, 2 & 5 mm 1, 2 & 5 cm NaPS 1 g Citrate buffer pH 6.0 75 gPU-film* Hydrogel 2: PVP K90 20 g none PEG-DMA 4 g 1, 2 & 5 mm 1, 2 & 5cm NaPS 1 g Propyleneglycol 10 g PU-film* Citrate buffer pH 6.0 65 g*Polyurethane film 30 μm in thickness

The final adhesive is sprayed at the adhesive side with vaccine preparedas described in example 1 to a level of 0.05 mg/cm². The material isdried, a new release liner is applied and patches are produced.

Example 14

Visualization of PosIntro Penetration of Stratum Corneum withFluorescence Microscopy

Human skin was obtained from a 37-year-old woman undergoing breastreduction surgery after informed consent (approved by local ethicalcommittee). The skin was cut into 8 mm biopsies using a biopsy-punch.Each explant was placed in an 8 mm diameter punching in an agarose gelcast in a cell culture insert for 12-well format. Acridin stainedPosIntro particles (10 μl, 300 μg/ml) were carefully applied as aspheric droplet on top of the epidermal layer (stratum corneum).Explants were cultured for 3 days at 37° C., 5% CO2, air-liquidinterface with Dulbeccos modified Eagles medium (DMEM) and 10% FCS. Thebiopsies was cryofixed in OCT (TissueTek) and 4 μM sections cut in acryomicrotome (Leica, Germany). The fluorescence signal (green) wasdetected in an Olympus BX60 fluorescence microscope using U-MWIB2wide-band interfence barrier filter.

FIG. 11A shows a section from the application site of PosIntro particles(100×). Basal membrane is indicated by the line.

FIG. 11B shows the same as FIG. 11 (200×) Arrowheads indicate areas withhigher fluorescence intensity, which might be attributable to LangerhansCells.

FIG. 11C shows a control section from the same slide as in FIGS. 11A andB, but distant from the application site of PosIntro particles. Theupper line indicates beginning of stratum corneum and the lower line thebasal membran

Example 15

Visualization of PosIntro Homing to Langerhans Cells in Epidermis

The experiment was repeated as described above and Langerhans cells werevisualized by a TRITC labelled CD1a antibody (Dako, Denmark). As shownin FIG. 12 the position of Langerhans cells in epidermis correlates verywell with areas of PosIntro accumulation.

FIG. 12A shows acridin stained PosIntro particles penetrating stratumcorneum. More intense stained areas are indicated by the arrows.

FIG. 12B shoes the same section as FIG. 12A with TRITC-CD1a indicatingthe presence of Langerhans cells at the position as depicted by thearrows in FIG. 12A.

Example 16

Visualization of PosIntro Penetration Through Stratum Corneum UsingConfocal Microscopy

Following 1 day of incubation explants with fluorescently labelledPosIntros applied on the epithelial side were cryo fixated in ice coldisopentane on dry ice in 99% ethanol. Frozen explants were divided intohalves and embedded in O.C.T. compound (TissueTek, Sakura) in cryomolds.

Explants were cryo sectioned 20 μm thick and the basement membranes werelabelled with primary mouse-anti-human Collagen IV a.b. (DAKO, M0785)overnight at 4° C. Next morning secundary labelling was performed withsecondary Alexa Flour 488 goat-anti-mouse a.b (Molecular Probes,A-11001). Finally slides were stained 1 minute with Meyers acidichaematoxylin.

Slides were microscopically evaluated with a Zeis LSM 310 confocalmicroscope equipped with a 488 nm and a 543 nm laser. Basement membranessecondary labelled with Alexa Flour 488 and Acridin Orange labelledPosIntros were excitated with the 488 nm laser. Haematoxylin contrastwas collected as a transmitted nonconfocal image with the 543 nm laser.

All confocal images were collected as single optical sections 8-15 μmbeneath the surface of the 20-30 μm cryosections using a 40× dryobjective, averaging ×2, time 8 with automatic brightness and contrastand additional manual contrast enhancement. Each picture represents themerger of two confocal single optical sections and one transmittednonconfocal image.

FIG. 13A shows confocal microscopy of skin biopsy after incubation withacridin PosIntro for 24 hours (40×). Presence of basal membrane isindicated by a FITC labelled collagen IV antibody.

FIG. 13B shows the same as FIG. 13A, but in 80×, indicating penetrationof stratum corneum and distribution of PosIntro particle aggregatesthrough epidermis.

FIG. 13C shows a biopsy as in FIGS. 13A and B without acridin PosIntro.

Example 17

Transfection of Fibroblast Cell Cultures with GFP-PosIntro

L929 fibroblasts were plated on 2-well chamberslides (Nunc, Roskilde,Denmark) and grown in medium containing 90% Dulbecco's modified Eaglesmedium and 10% fetal calf serum. Semi-confluent cells were transfected 3hours in serum free transfection-medium (Optimem-1, Gibco BRL,Invitrogen, UK) containing 2.5 μg/ml GFP-plasmid (gW1Z) and 10 μg/mlDC-PosIntro. Control cultures were transfected with GFP-plasmid alone orGFP-plasmid and 10 μg/ml Lipofectin (Gibco, Invitrogen, UK). Transfectedcells were cultured for 24 hours before evaluation by fluorescencemicroscopy.

As illustrated in FIG. 14, bright fluorescent GFP-expressing cultureswere obtained after transfection with DC-PosIntro and GFP-plasmid. Thetransfection efficiency was approx 1% (compared to 10-20% for lipofectintransfected cultures) however, the appearance of cells aftertransfection was much healthier with DC-PosIntro compared to Lipofectintreated cultures.

Example 18

Transdermal Hepatits B Surface Antigen Vaccination.

Three rabbits received a Poslintro formulated recombinant Hepatitis Bsurface antigen (HBs Ag). Three weeks after immunization venues bloodsamples were measured for HBs Ag specific antibodies by a competitionassay based on a modification of the bioMerieux, Vidas HBs antigen invitro diagnostic system.

Recombinant HBs antigen was obtained from Aldevron, USA. Antigen wasembedded intro Posintro by similar procedures as described in U.S.provisional application Ser. No. 60/308,609. However, prior to embedmentHBs antigen was coupled with palmetic acid in a 5:1 molar ration. Eachanimal received 30 μg antigen divided into three hydrogel 2 patchesprepared according to example 13 in a thickness of 2 mm and a diameterof 1 cm.

Twenty-four hours after immunization animals were shaved thoroughly andwashed at the site of immunization. Immunization patches were placed atthree different positions at the back of each animal and supported by anadditional layer of Comfeel® transparent dressing, Coloplast, Denmark.As far as possible the patches were allowed to sit for 24 h, however dueto animal activity patches were partly released form the skin duringthis period.

Three weeks after immunization venous blood samples were drawn andantibody titer specific to HBs Ag measured. Antibodies were measured bymixing serial dilutions of rabbit serum with a constant amount ofrecombinant HBs Ag in normal human plasma (S1 standard from bioMerieux,Vidas HBs antigen kit). The mixtures were measured after 10 min.incubation by bioMerieux, HBs antigen diagnostic kits in the Vidassystem. Antibody titeres are given as the highest point of dilutionwhere the detected level of HBs Ag are reduced to less than 50% comparedto the addition of normal negative serum. All animals were testednegative for antibodies specific to HBs Ag prior to immunization. TABLE2 Test Test Test Titer prior to value value value Rabbit no.immunization 10⁰ 10² 10⁴ Titer A1 <1 0.18 1.03 n.d. >10⁰ A2 <1 0.03 0.411.04 >10² A3 <1 0.02 0.15 1.07 >10²Titers of Hepatitis B Surface antigen (HBs Ag). Antibody titers aregiven as the highest dilution of serum, 100-fold dilutions, at which thespecific detection of HBs antigen is inhibited at least 50%. Test valuesare arbitrary units with a maximum value of 1.14.Table 2

Titers of Hepatitis B Surface antigen (HBs Ag). Antibody titers aregiven as the highest dilution of serum, 100-fold dilutions, at which thespecific detection of HBs antigen is inhibited at least 50%. Test valuesare arbitrary units with a maximum value of 1.14.

Example 19

Transdermal Tetanus Vaccination

Groups of rabbits (5 animals) received a transdermal tetanus vaccineformulated with saponin or embedded into Posintro particles asillustrated in Example 2 and in a hydrogel 2 patch dimensioned as givenin example 13, respectively. Blood samples were measured by ELISA 3weeks after immunization and antibody titers determined. Titers aregiven in FIG. 15 as the highest dilution (5-fold) at which theabsorbance reading is reduced below 50% of the titer at 5⁰ dilutions.

1. Construct for transdermal delivery of at least one immunogen to anindividual comprising a) said at least one immunogen b) an occlusionvehicle and c) an immunogen delivery system wherein the immunogendelivery system is a complex comprising: i) at least one first steroland/or at least one second sterol, wherein the at least one secondsterol is capable of contacting a genetic determinant by means of aninteraction selected from an electrostatic interaction and a hydrophobicinteraction, and wherein the at least one first sterol and/or the atleast one second sterol is capable of forming a complex with at leastone first saponin and/or at least one second saponin, and ii) at leastone first saponin and/or at least one second saponin, wherein the atleast one second saponin is capable of contacting a genetic determinantby means of an interaction selected from an electrostatic interactionand a hydrophobic interaction, and wherein the at least one firstsaponin and/or the at least one second saponin is capable of forming acomplex with at least one first sterol and/or at least one secondsterol, and optionally iii) at least one contacting group for contactinga genetic determinant by means of an interaction selected from anelectrostatic interaction and a hydrophobic interaction, with theproviso that the at least one contacting group is present when no secondsterol is present in the complex and further optionally iv) at least onelipophilic moiety.
 2. Construct according to claim 1, wherein theocclusion vehicle is a pressure sensitive adhesive.
 3. (canceled) 4.Construct according to claim 1, wherein the transdermal deliveryincludes delivery through a skin surface or through a mucous membranetissue.
 5. Construct according to claim 1, wherein the occlusion vehicleis a absorbing pressure sensitive adhesive.
 6. Construct according toclaim 1, wherein the occlusion vehicle is a hydrocolloid adhesive. 7.Construct according to claim 1, wherein the occlusion vehicle is ahydrogel adhesive.
 8. Construct according to claim 1, wherein theocclusion vehicle is a cross-linked hydrogel adhesive.
 9. Constructaccording to claim 1, wherein the immunogen and the immunogen deliverysystem is distributed preferably homogenously in the occlusion vehicle.10. Construct according to claim 1, wherein the immunogen and theimmunogen delivery system is distributed on the surface of the occlusionvehicle.
 11. Construct according to claim 1, wherein the occlusionvehicle is a non-adherent occlusion vehicle, and further comprising asecondary adhesive, being separated from the vehicle, for skin fixation.12. Construct according to claim 11, wherein the occlusion vehicle isdried or lyophilised and contains a carrier comprising a hydrophilicpolymer substance or a grease like composition.
 13. Construct accordingto claim 1, wherein the occlusion vehicle or the secondary adhesive is acovering, such as a pad, a patch, a dressing or the like.
 14. Constructaccording to claim 12 further comprising a reservoir of water or otherappropriate solvent/diluent.
 15. Construct according to claim 14,wherein the water reservoir can be broken and the water orsolvent/diluent can be absorbed in the occlusion vehicle.
 16. Constructaccording to claim 1 further comprising a rate controlling membrane. 17.Construct according to claim 1, wherein the immunogen and/or theimmunogen delivery system is separated from each other.
 18. Constructaccording to claim 1 further comprising an enhancer for transdermal drugdelivery.
 19. Construct according to claim 1, wherein the at least oneimmunogen is selected in such a way that the induced immunologicalresponse is directed against one or more antigens.
 20. Constructaccording to claim 19, wherein said one or more antigens are derivedfrom a microorganism, preferably a pathogenic microorganism, such as avirus, a bacteria, a parasite and/or a fungus, or from a non-microbialorganism, e.g. from an animal, such as a vertebrate.
 21. Constructaccording to claim 19, wherein the immunogen and/or antigen are derivedfrom a virus.
 22. Construct according to claim 21, wherein said one ormore antigens are synthetic antigens, antigens derived from saidindividual or antigens derived from any species.
 23. Construct accordingto claim 19, wherein the at least one immunogen is selected in such away that the induced immunological response confers protection in saidindividual against a pathogenic microorganism which said antigen orantigens are part of.
 24. Construct according to claim 19, wherein theat least one immunogen is selected in such a way that the inducedimmunological response may act upon subsequent exposure of theindividual to said pathogenic microorganism.
 25. Construct according toclaim 19, wherein the at least one immunogen is selected in such a waythat the induced immunological response is directed against a pathogeniccomponent produced by said pathogenic microorganism during infection ofsaid individual, e.g. bacterial toxins, such as tetanus toxin. 26.Construct according to claim 1, wherein the immunogen and/or antigencomprise or consist of i) one or more identical or differentpolypeptides and/or peptides, which polypeptides and/or peptidesoptionally comprise posttranslational modifications, ii) one or moreidentical or different lipopeptides, such as polypeptides and/orpeptides chemically linked to a lipid group, iii) one or more identicalor different nucleic acid sequence or sequences, which may encodepolypeptides and/or peptides, or iv) one or more identical or differentpolysaccharides and/or oligosaccharides, or combinations thereof, andwherein the immunogen and/or antigen may further be processed intofragments.
 27. Construct according to claim 1, wherein the immunogen andthe immunogen delivery system is comprised within a vaccine formulation.28. (canceled)
 29. Process for the preparation of a construct accordingto claim 1, comprising the steps of introducing the immunogen and theimmunogen delivery system, which are optionally comprised within avaccine formulation, into the matrix of the occlusion vehicle or on itssurface by dispersion or soaking in a solution of the vehicle or byapplying to its surface, and optionally sterilising and/or drying and/orseal packaging the construct.
 30. Process according to claim 29 furthercomprising the step of drying or lyophilisation or the immunogen and theimmunogen delivery system before introducing into the vehicle. 31.Process according to claim 29 further comprising the step of adding oneor more enhancers for transdermal drug delivery and/or one or moreplasticizers.
 32. Construct according to claim 1, having one or morecompartments.
 33. Construct according to claim 32 having at least twocompartments, wherein a first compartment comprises a lyophilised padcomprising the immunogen and the immunogen delivery system and a secondcompartment comprises water or other appropriate solvent/diluent. 34.Construct according to claim 1 comprising at least two separatecomponents.
 35. Method for generating an immunological response in anindividual wherein said individual is treated transdermal with aconstruct according to claim
 1. 36. Method for treating or preventing acondition of illness in an individual, e.g. a disease caused byinfection of said individual by a pathogenic microorganism, wherein saidindividual is treated transdermal with a construct according to claim 1.37. Method for vaccination of an individual wherein said individual istreated transdermal with a construct according to claim 1.